Enhanced Anticancer Activity of Gemcitabine in Combination with Noscapine via Antiangiogenic and Apoptotic Pathway against Non-Small Cell Lung Cancer

BACKGROUND:The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos) and Gemcitabine (Gem) combination (NGC) against non-small cell lung cancer (NSCLC) and to elucidate the underlying mechanism of action. METHODS:Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues. RESULTS:The CI values (<0.59) were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups. CONCLUSION:Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer

Identification of a butyrophenone analog as a potential atypical antipsychotic agent: 4-[4-(4-Chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analog of haloperidol, 4-[4-(4-Chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (Compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED50 value

Antitumor Activity of Noscapine in Combination with Doxorubicin in Triple Negative Breast Cancer

The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC).TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues. values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups.Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC

Immunohistochemical staining of MDA-MB-231 tumor tissues for (A) VEGF expression.

<p>Tumor sections were stained using the ABC staining kit as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017733#s2" target="_blank">Materials and Methods</a>. Cells showing positive VEGF expression are stained brown. Original magnification ×40. (Micron bar = 100 µm). Immunohistochemical staining of MDA-MB-231 tumor tissues for (<b>B</b>) CD31 expression. Tumor angiogenesis was assessed by immunohistochemical staining with anti-CD31 antibody (brown) on paraffin-embedded sections. Original magnification ×40. (Micron bar = 100 µm). (<b>C</b>) Quantitation of apoptotic cells from VEGF staining. (<b>D</b>) Assessment of microvessel density. Microvessel density (MVD) was calculated by selecting three most vascularised areas of the tumour (‘hot spots’) and mean values obtained by counting vessels. A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen. Microvessel counts were performed at ×400 (×40 objective lens and ×10 ocular lens; 0.74 mm² per field). The MVD was significantly different between the control group and treated groups in sequential analysis; **, <i>P</i><0.01;* <i>P</i><0.05 relative to control.</p

Western blotting of tumor tissue lysates to determine expressions apoptosis-related proteins (A) expression of NF-kβ, IKBα, P-IKBα, Bax, Bcl2, caspase 3, cleaved caspase 3, activated caspase 8 and activated caspase 9 proteins in tumor lysates by western blotting and (B) quantitation of apoptotic protein expression.

<p>Tumor tissue lysates harvested tumor tissues from control-untreated and treated groups were analyzed by western blotting for protein expressions. Lane 1 = control; Lane 2 = Noscapine 150 mg/kg/day; Lane 3 = Noscapine 300 mg/kg/day; Lane 4 = Noscapine 450 mg/kg/day; Lane 5 = Noscapine 550 mg/kg/day; Lane 6 = Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule; Lane 7 = Combination (Noscapine 300 mg/kg/day+Doxorubicin1.5 mg/kg i.v. bolus, q3d×7 schedule). Similar results were observed in triplicate experiments. Protein expression levels (relative to β-actin) were determined. Mean ± SE for three replicate determinations. One-way ANOVA followed by post Tukey test was used for statistical analysis. <i>P</i><0.01 (*, significantly different from untreated controls; ^**, significantly different from Noscapine and Doxorubicin single treatments).</p

Combination Index (CI) values of the interaction between Nos with Dox against human MDA-MB-231 and MDA-MB-468 TNBC cells.

<p>The human lung cancer cell lines MDA-MB-231 and MDA-MB-468 breast cancer cells were obtained from American Type Culture Collection (Rockville, MD). Different concentrations of Nos were employed to study the effect on IC50 of Dox. Variable ratios of drug concentrations and mutually non-exclusive equations were used to determine the CI. The CI values represent mean of four experiments. CI>1.3: antagonism; CI 1.1–1.3: moderate antagonism; CI 0.9–1.1: additive effect; CI 0.8–0.9: slight synergism; CI 0.6–0.8: moderate synergism; CI 0.4–0.6: synergism; CI 0.2–0.4: strong synergism.</p

Fluorescence Micrographs of cells stained with rhodamine and DAPI after 72 h (A) with Doxorubicin 0.4 µg/ml , Noscapine 30 µM, and, Noscapine and Doxorubicin combination in MDA-MB-231 cells and (B) Quantitation of apoptotic MDA-MB-231 cells from TUNEL assay.

<p>DNA fragmentation indicated by positive staining (red) and nuclear condensation indicated by DAPI nuclear staining (blue). Micron bar = 100 µm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean+SD (N = 6). One-way ANOVA followed by post Tukey test was used for statistical analysis to compare control and treated groups. * <i>P</i><0.01; all treatments significantly different from control and ** <i>P</i><0.01; significantly different from Noscapine and Doxorubicin single treatments.</p

Progression profile of tumor growth kinetics of in-vivo antitumor effect of different doses of Noscapine alone (A) and in combination with Doxorubicin (B) on human MDA-MB-231 tumor xenograft model (tumor volumes, mm³ ± SEM), and measurement of body weight following Noscapine alone (C) and combination with Doxorubicin (D).

<p>Female nude mice with xenograft MDA-MB-231 tumor tumors received various treatments for 38 days starting on day 7 post tumor implantation. The mice were treated with Noscapine (150–550 mg/kg/day), Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule, and Noscapine 300 mg/kg/day+Doxorubicin 1.5 mg/kg i.v. bolus, q3d×7 schedule. Control group received vehicle only. Statistical significance of the difference in tumor volume of treatment groups compared with control. <i>P</i><0.01 (*, significantly different from untreated controls; ^**, significantly different from Noscapine and Doxorubicin single treatments). Data presented are means and SE (n = 8). This experiment was repeated twice.</p